Light‐controlled scaffold‐ and serum‐free hard palatal‐derived mesenchymal stem cell aggregates for bone regeneration

Abstract Cell aggregates that mimic in vivo cell–cell interactions are promising and powerful tools for tissue engineering. This study isolated a new, easily obtained, population of mesenchymal stem cells (MSCs) from rat hard palates named hard palatal‐derived mesenchymal stem cells (PMSCs). The PMSCs were positive for CD90, CD44, and CD29 and negative for CD34, CD45, and CD146. They exhibited clonogenicity, self‐renewal, migration, and multipotent differentiation capacities. Furthermore, this study fabricated scaffold‐free 3D aggregates using light‐controlled cell sheet technology and a serum‐free method. PMSC aggregates were successfully constructed with good viability. Transplantation of the PMSC aggregates and the PMSC aggregate‐implant complexes significantly enhanced bone formation and implant osseointegration in vivo, respectively. This new cell resource is easy to obtain and provides an alternative strategy for tissue engineering and regenerative medicine.

harmful materials. Previous studies have reported that light-controlled cell sheet technology combined with vitamin C could fabricate cell sheets in a convenient and safe manner. [24][25][26] Furthermore, light is an easy-to-control source, saving time and increasing efficiency during experiments. 27 However, few investigations have reported the successful harvest of cell aggregates under light illumination. In this study, we used a light-controlled method to fabricate cell aggregates.
Cell aggregates can mimic native cellular microenvironments in vivo. 28 In particular, MSCs isolated from hard palates are promising cell sources for in vivo transplantation owing to their advantages of easy isolation, multipotent differentiation, and fast proliferation.
Cell-cell/cell-extracellular matrix (ECM) interactions have improved cell viability compared with single cells. 29 Cell aggregates could limit the immobility of cells at the site of the defect and improve transplantation efficiency. 30 Moreover, they have better osteogenic differentiation potential than cell sheets. 31 These factors may contribute to the improved bone healing ability of cell aggregates.
Therefore, we assumed that PMSC aggregates could accelerate bone regeneration in vivo.
The composition of serum is complex and has not been completely determined. It contains a large number of microorganisms that may have adverse effects on cell growth. 32 Moreover, the storage life of serum is limited, and there is high variability between batches. 33 Therefore, it is not ideal for clinical applications. Recently, it was found that MSCs could form cell aggregates in serum-free culture medium. 34,35 This procedure had several advantages, such as a low probability of microbiological contamination or transmission of animal diseases to humans, low cost, and high reproducibility. 23,36 Cell sheet technology combined with serum-free culture may provide a novel method for reliable clinical applications of cell aggregates to guarantee well-defined compositions with a low risk of contamination.
The aims of this study were to isolate rat PMSCs and evaluate the feasibility of harvesting scaffold-free and serum-free cell aggregates via light-controlled cell sheet technology. Furthermore, we aimed to evaluate the bone regeneration capacity of PMSC aggregates.

| Characteristics of the PMSCs
PMSCs originated from the lamina propria layer of the hard palate and were close to the basement membrane ( Figure 2a). In this study, we F I G U R E 1 Wound healing processes of palatal-derived mesenchymal stem cells (PMSCs) and adipose-derived mesenchymal stem cells (AMSCs) harvesting. (a) A long strip of mucosa of 1.5 mm Â 3 mm was removed from the hard palate. The wound showed a pink, healthy healing appearance without infection from the second day after surgery. (b) Subcutaneous adipose tissue was removed from the inguinal region and the harvesting wound was intermittently sutured. The healing time of the wound was approximately 8 days. During this period, the wound was prone to dehiscence. (c) The healing time of PMSCs harvesting was significantly shorter than that of AMSCs. (d) All the hard palatal mucosa wounds were healed without incident, while the adipose tissue harvesting sites had a high wound dehiscence rate. *: p <0.05 isolated PMSCs and turned them into cell sheets and aggregates, which were easily and safely harvested under light activation ( Figure 2b). As Figure 2c shows, spindle-shaped cells migrated from the hard palate tissue, congregated and finally formed cell colonies ( Figure 2d). In addition, the culturing time decreased from passage 0 to passage 3, while after passage 3, the culture time gradually To learn more about the characteristics of PMSCs, several experiments were performed, comparing PMSCs with other three kinds of cells. Cell scratch assays showed that the two cell sources derived from oral mucosa, namely PMSCs and gingival-derived mesenchymal stem cells (GMSCs), had similar high migration ability (Figure 4a). The wound closure rates were 24.5 ± 0.8% and 21.0 ± 3.3%, respectively.
On the contrary, bone mesenchymal stem cells (BMSCs) exhibited a significantly lower migration ability with a wound closure rate of 6.9 ± 6.7% (p <0.05). To evaluate the cellular responses under different circumstances, cells were subjected to osteogenic induction and tumor necrosis factor-α (TNF-α) stimulation. RT-qPCR assays showed that the expression levels of osteogenic (BMP2, low-density lipoprotein receptor-related protein 5 [LRP5], and β-catenin) were significantly increased in PMSCs (Figure 4b). BMSCs exerted the most positive response to osteogenic induction with high levels of related gene expression. On the contrary, GMSCs were not able to be induced by osteogenic medium. Finally, the expression levels of genes associated with inflammation were estimated ( Figure 4c). AMSCs, BMSCs, and GMSCs showed noticeable changes in the mRNA expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), or inducible nitric oxide synthase (iNOS), while PMSCs seemed to be more insensitive to inflammatory induction. GMSC group showed significant increase in the expression of transforming growth factor-β (TGFβ). Except for AMSCs, all cell sources showed remarkable decrease of interferon γ (IFNγ).

| Characteristics of the PMSC sheets and PMSC aggregates
After culture on nanodot platforms for 5 days, PMSCs proliferated and formed intact cell sheets (Figure 5a). The migrating and reattaching capacities of the PMSC sheets were examined. As  value than the tibias with the SLA implant (p <0.05, Figure 8c).
In the rat tibial defect model, micro-CT analysis indicated that after 4 and 8 weeks of healing, BV/TV and Tb.N of the tibias with the PMSC aggregates were significantly higher than those of the control group, and Tb.Sp was significantly lower. As seen in the 3D reconstruction images, the injured tibias were much stronger and more intact in the PMSC aggregate group, representing a more satisfying healing outcome (Figure 8d).

| DISCUSSION
Exploring a new resource of MSCs with advantages of easy isolation and rapid healing is a significant research issue in tissue engineering and regenerative medicine. Oral mucosa might be a simple and alternative MSCs resource due to its strong regenerative capacity. 37,38 Recently, the potential role of MSCs in enhancing bone formation in the clinics has been proven. 39 In this study, we reported the isolation and culture of rat PMSCs with rapid wound healing and no risk of wound dehiscence for the first time. We investigated the fabricating strategy of PMSC aggregates through light-controlled and serum-free method, and evaluated their bone regeneration ability in vivo.
Our study is consistent with the previous studies that MSCs have played a leading role in regenerative medicine due to their selfrenewal ability and potential to differentiate into various cell types. [40][41][42][43] To the best of our knowledge, this is the first study to show MSCs could be isolated from postnatal rat hard palates. Previous studies have reported embryo palatal MSCs, [44][45][46] palatal periosteum MSCs, 18 and MSCs from adipose tissue of the hard palate. 17 The oral mucosa, including the hard palate mucosa, is a rapidly dividing tissue with great regeneration capacity. 47,48 Compared to MSCs derived from other organs, such as bone marrow MSCs, PMSCs showed advantages with easier and less invasive harvesting procedures. Therefore, they may present great potential in future clinical applications. Trilineage differentiation analysis, proliferative capacity, and cell markers confirmed isolation of MSCs, 49-52 strongly supporting that PMSCs represent a MSC population. By comparing with traditional cell sources including AMSCs, BMSCs and GMSCs, we provided an initial impression of PMSCs. Cell scratch assays indicated excellent cell migration ability of these oral mucosa-derived cells. This can be an advantage as cell migration is essential for various biological functions. 53 Considering the potential application in osseointegration acceleration and bone defect healing, PMSCs were evaluated for its osteogenic capacity and tolerance to inflammation. In consistence with the results of trilineage differentiation assay, PMSCs showed positive response to osteogenic induction despite the degree of increase in osteogenic genes was relatively lower than BMSCs. As for TNF-α stimulation, though the mRNA expression trends of proinflammatory and anti-inflammatory factors varied among the four kinds of cells, we might suppose that PMSCs were relatively insensitive to inflammatory challenge. This is also an advantage since osseointegration and bone healing can be impaired by inflammation. 54,55 However, the differences between PMSCs and MSCs from other tissues remained to be explored in the future study. Besides, whether PMSCs could be used for fabrication of cell aggregates are not clear.
Cell aggregates mimic cell-to-cell interactions and cell-to-ECM interactions that more closely reflect characteristics in native tissues.
They have been widely applied in drug screening, 56,57 regenerative medicine, 19 and tumor research. 58 Previous studies have reported that cells could form cell aggregates using foreign materials with culture medium containing serum, 22 hindering their large-scale implication in the clinic. Therefore, we developed light-controlled serumfree method for harvesting cell aggregates in this study. Light illumination was able to induce changes in the wettability or water content of the culturing surfaces for cells. 25,59 These changes could lead to the conformational changes in or the release of the adhesive proteins or collagens, which contributed to cell detachment. In addition,  60 and then the potential change induced by electron accumulation would also manipulate protein release. 61 When adopting light illumination for cell harvesting, the wavelength and light dose (depending on power density and irradiation time) must be defined first. 59 To be specific, researchers have to consider biocompatibility and harvest efficiency since shorter-wavelength light with a higher energy also exerts greater harm to cell viability. Generally, preliminary experiments are conducted to select a light source suitable for the cell culturing material, and to determine other parameters such as the shortest irradiation time for the most efficient harvesting. Previous studies published by our team had established a protocol based on a nanostructured TiO 2 surface, 365 nm UV light, and irradiation duration of 30 min. 62 The present study demonstrated that PMSCs could form cell aggregates and could be harvested through light activation. The cell aggregates were characterized by a high cell density and good viability. 63,64 Although the growth rate of isolated PMSCs would be slowed down after serum-free culture, this strategy was only applied after cell sheet formation for the fabrication of cell aggregates, which means cell proliferation was not truly important at this point. Furthermore, they could preserve ECM and biological signals, which might facilitate the bone regeneration process. 65,66 Cell populations displaying high cadherin expression were found in the interior, whereas cells with high integrin expression were found in the exterior of the aggregates. 31,67 In this study, immunofluorescence staining demonstrated that PMSC aggregates contained a large amount of MSCs and abundant ECM. Furthermore, a live-dead staining assay indicated that PMSC aggregates had good cell viability, which was also consistent with previous studies. 29,68 We could also infer that the serum-free culturing and light-induced harvesting process of PMSC aggregates in this study was safe and effective.
Cell aggregates have various advantages. First, presented stronger anti-inflammatory effects and increased angiogenesis potential, 69,70 while immunomodulatory-related gene expression was lower. 71 Second, great osteogenic capacity of cell aggregates was confirmed by a number of researches [72][73][74] and our in vivo studies. Cytochemical analysis, gene expression quantification, and protein expression quantification showed that MSC aggregates were associated with increased ALP activity and higher levels of expression of osteogenic markers, including osteocalcin, ALP, Runx2, collagen I, and BMPs. [74][75][76] Related signaling pathways might include the Wnt/βcatenin and BMP-Smad pathways, as indicated by more significant upregulation of the p-Smad1/5, p-p38, phospho-extracellular signalregulated kinase (p-ERK), β-catenin, and secreted frizzled-related protein 3 (SFRP3) that was detected in the cell aggregates. 77,78 Better and faster osseointegration around titanium implants modified with PMSC aggregates was also observed in our study.
Osseointegration could be influenced by implant surface characteristics, including roughness and wettability. 79,80 Since this process requires the migration, proliferation and differentiation of osteogenic cells, physical or chemical surface modification might not be effective enough to promote osseointegration. 81 Previous studies have reported enhanced osseointegration with various cell sheet-modified implants 62,82 and ECM sheet-modified implants. 83 In this study, we managed to fabricate PMSC aggregates-implant complexes that successfully enhanced osseointegration.
Therefore, PMSC aggregates could be novel biomaterials for bone regeneration. Recently, cell aggregates have been used to deliver drugs 84 and genes. 85 Previous studies have succeeded in promoting bone regeneration with genetically modified dissociated cells and cell sheets. 86 In the future, PMSC aggregates may serve as a potential drug and gene delivery vehicle to enhance their osteogenic capacity. the subcutaneous adipose tissues were harvested from the inguinal region of the rats and also cultured in basal medium. The wounds created by adipose tissue harvesting were carefully sutured. The healing processes of PMSCs and AMSCs harvesting sites were closely observed and recorded from the second day after surgery until the tenth day. Both tissues were incubated in an atmosphere composed of 95% humidity and 5% CO 2 at 37 C. After the first 24 h, the culture medium was changed and then replaced every 3 days. After 8 days, the remaining tissue debris was removed, and the adherent cells were digested and passaged. The medium was replaced every 2 days and passaging occurred every 4-5 days. BMSCs were isolated and cultured according to our previous protocols. 70 The shapes and quantities of cells were visualized under a microscope (Zeiss, Germany).
PMSC aggregates were observed under a phase-contrast microscope (Zeiss, Germany).

| Cell scratch assay
Rat PMSCs, GMSCs, BMSCs, and AMSCs at passage 3 were seeded in 12-well plates at a density of 1 Â 10 5 cells/cm 2 for cell culture, and the culture medium was changed every 2 days. After the cell mono-

| Inflammatory cytokines release under inflammatory environment
Rat PMSCs, GMSCs, BMSCs, and AMSCs at passage 3 were seeded in 6-well plates at a density of 1 Â 10 5 cells/cm 2 and cultured with α-MEM for 24 h until the cells adhered. Then, the basic medium was replaced with medium containing TNF-α (10602-HNAE, Sinobiological, China) of 50 ng/ml. After 3 days of incubation, total RNA was purified, reverse transcription to cDNA and RT-qPCR assays were conducted. The primers for the targeted genes were as follows: IL-1β, The total energy was 2520 mJ/cm 2 (<safe energy 7500 mJ/cm 2 ) 87 after illumination for 30 min. Subsequently, to promote cell aggregate formation, the culture medium was changed from α-MEM to serumfree medium. The PMSC sheets self-assembled into PMSC aggregates after culture in serum-free medium on a TiO 2 nanodot platform for 24 h. Finally, PMSC aggregates were harvested through 365 nm UV light irradiation for 30 min.

| Readhesion assay
A total of 1 Â 10 5 cells were seeded in 12-well plates and cultured with α-MEM. After 5 days of culturing, PMSC sheets were harvested through irradiation with 365 nm light for 30 min and reseeded in a new 12-well plate. Subsequently, the culture medium was changed every 2 days. Extreme care was taken to avoid movement or floating of the sheets. After 1, 2, 3, and 4 days, adhesion of the PMSC sheets to the plates and the cell growth around the sheets were recorded using a phase-contrast microscope (Zeiss, Germany).

| Live-dead staining of PMSC sheets and PMSC aggregates
To evaluate the cell viability of the PMSC sheets and PMSC aggregates, a live-dead staining assay was performed. Briefly, PMSC sheets and PMSC aggregates were stained with calcium for 30 min and PI for 10 min at 37 C before and after illumination with 365 nm light. Cell morphology was recorded using an inverted fluorescence microscope (IX81, Olympus, Japan). Cell sheets were immersed in 4% PFA for 15 min as a negative control.

| Immunofluorescence of PMSC sheets and PMSC aggregates
The cell sheets and cell aggregates were harvested for immunofluorescence to observe their composition and structure. They were incubated with antibodies (fibronectin and CD90) for 15 h and phalloidin for 2 h. Cell morphology was observed using an inverted fluorescence microscope (IX81, Olympus, Japan).

| Statistical analysis
Statistical analysis was conducted using two-tailed unpaired Student's t-test to compare two groups or by one-way ANOVA with Tukey-Kramer post hoc test to compare three groups. p <.05 was considered to be significant.